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X/1/2019
InterdIscIplInarIa archaeologIca
natural scIences In archaeology
homepage: http://www.iansa.eu
Looking Beyond the Surface: Use of High Resolution X-Ray Computed
Tomography on Archaeobotanical Remains
Charlene Murphy
a
, Dorian Q. Fuller
a,b*
, Chris Stevens
a,c
, Tom Gregory
a
, Fabio Silva
d,e
, Rita Dal
Martello
a
, Jixiang Song
f
, Andrew J. Bodey
g
, Christoph Rau
g
a
UCL, Institute of Archaeology, 31–34 Gordon Square, London, WC1H 0PY, United Kingdom
b
School of Cultural Heritage, Northwest University, Xian, 710069, China
c
School of Archaeology and Museology, Peking University, 100871, China
d
Department of Archaeology, Anthropology and Forensic Science, Bournemouth University, Fern Barrow, Poole, Dorset, BH12 5BB, United Kingdom
e
Faculty of Humanities and Performing Arts, University of Wales Trinity Saint David, Lampeter Campus, Ceredigion, SA48 7ED, United Kingdom
f
Department of Archaeology, Center for Archaeological Science, Sichuan University, Chengdu, China
g
Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, OX11 0DE, United Kingdom
1. Introduction
High Resolution X-Ray Computed Tomography (HRXCT)
imaging ofers a powerful 3-dimensional, non-destructive
and non-invasive diagnostic tool to image the external and
internal properties of a range of specimens of interest (Friis
et al.
, 2014). HRXCT ofers new possibilities in terms of the
research questions which may be asked of fragile and valuable
archaeological and specifcally archaeobotanical material. This
paper will discuss the methods and results of the successful use
of HRXCT to image preserved archaeobotanical specimens
of horsegram (
Macrotyloma uniforum
) from diferent time
periods ranging from the Neolithic (4,000 BP) to modern
specimens (present day) from South Asia, and additional taxa
including lentil (
Lens culinaris
), goosefoot (
Chenopodium
sp.), and soybean (
Glycine max
).
HRXCT has grown in popularity recently as it has become
more user friendly – with a broad range of disciplines
employing this imaging technique, including: material
sciences (Guo
et al.
, 2017), art conservation (Cotte, 2016),
conservation (Wilson
et al.
, 2017), heritage studies (Bertrand
et al.
, 2012), paleobotany (DeVore, Kenrick, Pigg, 2006;
Friis
et al.
, 2007; 2014; Scott
et al.
, 2009), plant sciences
(McElrone
et al.
, 2013; Staedler
et al.
, 2013), archaeological
artefacts (Mocella
et al.
, 2015; Bukreeva
et al.
, 2016),
archaeobotanical material (Murphy and Fuller, 2017; Zong
et al.
, 2017), soil micromorphology (O´Donnell
et al.
, 2007)
and sedimentology (Appoloni
et al.
, 2007).
HRXCT is a very efective method for a large range of
disparate disciplines as it encompasses a wide variety of
Volume X ● Issue 1/2019 ● Pages 7–18
*Corresponding author. E-mail: d.fuller@ucl.ac.uk
ArTICLE INFO
Article history:
Received: 2
nd
May 2018
Accepted: 9
th
May 2019
DOI: http://dx.doi.org/ 10.24916/iansa.2019.1.1
Key words:
High-Resolution X-Ray Computed Tomography
(HRXCT)
Synchrotron
morphometrics
imaging
archaeobotany
ABSTrACT
High Resolution X-Ray Computed Tomography (HRXCT) ofers a powerful 3-dimensional, non-
destructive and non-invasive diagnostic tool for imaging the external and internal structures of a range
of specimens of interest including archaeobotanical remains. HRXCT ofers new possibilities in terms
of the research questions which may be asked of fragile and valuable archaeological and specifcally
archaeobotanical material. This technology, although currently somewhat limited in terms of time and
access to beamtimes at National Synchrotrons, requires simple, non-destructive preparation of samples
and produces exciting results. Based upon two rounds of successful work, we believe that this new
methodology has wider implications and utility for advancing the feld of imaging, and investigating
aspects of plant domestication such as internal anatomical changes.
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Looking Beyond the Surface: Use of High Resolution X-Ray Computed Tomography on Archaeobotanical Remains
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advanced microscopy and imaging techniques, which can
be adapted to the heterogeneous and complex structures
of the materials imaged (Bertrand
et al.
, 2012; Cnudde
and Boone, 2013). Resolutions can be a little better than a
micron with microtomography, and extend to nanometers
with nanotomographic methods.
For our research purposes, we looked at both charred and
modern seeds. We started with a 15 KeV monochromatic
beam, but progressed to using a fltered pink (polychromatic)
beam for an improved high signal to noise ratio, which
provided consistent and excellent image contrast for most plant
applications (McElrone
et al.
, 2013; Murphy and Fuller, 2017).
1.1 Synchrotron technology
HRXCT with a synchrotron employs the same principles as
current medical CAT-scanning to generate three-dimensional
images from two dimensional projections taken at diferent
orientations of the specimen. HRXCT uses energies that are
high enough to penetrate and study the internal structures
of the specimens (Bird
et al.
, 2008; Pantos, 2005, p.199).
Synchrotron radiation is made up of extremely bright light.
This type of radiation is naturally emitted by cosmic sources,
but it can also be generated at synchrotron facilities such as
Diamond Light Source in the UK (Figure 1) (Bertrand
et al.
,
2012). Synchrotron radiation is produced when electrons are
sped up to extremely high velocities by a linear accelerator
and sent into orbit within a storage ring. Magnets, placed
at various locations throughout the synchrotron storage
ring, regularly bend the path of the accelerated particles
as they circulate, generating synchrotron radiation (Winick,
1994; Greene, 2016) – Figure 2. The synchrotron radiation
is produced with a high degree of parallelism, ranging
Figure 1.
Aerial view of the UK
Synchrotron. Image courtesy of Diamond
Light Source.
Figure 2.
View of the inside of the ring
at the UK Synchrotron. Image courtesy of
Diamond Light Source.
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Looking Beyond the Surface: Use of High Resolution X-Ray Computed Tomography on Archaeobotanical Remains
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Figure 3.
Walkway view of the signal-to-
noise ring at the UK Synchrotron. Image
courtesy of Diamond Light Source.
Figure 4.
View of the Diamond-Manchester
Imaging Branchline I13-2. Measuring 250
metres in length, it is the longest beamline
at Diamond Light Source, the UK’s
Synchrotron in Oxfordshire, UK. Image
courtesy of Diamond Light Source.
over energy levels at one extreme as low as infrared light
to the so-called hard X-rays at the other (Nakai, 2005,
pp.183–184). Beams generated from a synchrotron ring are
four to ten orders of magnitude brighter than conventional
sealed-tube X-ray emitters (Pantos, 2005, p.199; Greene,
2016). This enables faster imaging with a higher signal-to-
noise ratio.
Based on the polygonal ring structure of a synchrotron
facility, the synchrotron radiation is sent at long oblique
angles, and transmitted down beamlines that terminate in
experimental stations (Figures 1 and 2). Energy profles
and fux vary between beamlines, allowing for diferent
types of experiments. Indeed, the advantage of synchrotron-
based techniques is the brightness attained and the range
of energies that allow a wide spectrum of materials to be
imaged (Bertrand
et al.
, 2012). Also, the partial coherence
enabled by a long beamline such as the Diamond-Manchester
Imaging Branchline I13-2 enables phase-contrast imaging;
this is particularly useful for heritage, archaeological and
material science felds that often possess a range of diferent
elements within one specimen are often composed of low
attenuating elements.
1.2 History of the technique and current trends
The potential of HRXCT as a non-destructive imaging
technique for ancient and historical materials was recognized
as early as the mid-1980s, with progress being made from
2000 onwards as major contributions from a narrow set
of expert users expanded to include a wider range of non-
specialized users from diferent felds (Bertrand
et al.
,
2012). As a result of this interdisciplinary collaboration,
novel analytical strategies are becoming increasingly
common as developments in advanced optics, detectors and
other instrumental developments coincide (Bertrand
et al.
,
2012). The cultural heritage and scientifc archaeological
communities have used, for the most part, the micro-focused
spectroscopy capabilities, using a reasonably low beam
divergence of synchrotron beamlines for either precise spot
analyses or in raster-scanning mode to map areas of the
samples being imaged (Bertrand
et al.
, 2012). This trend has
also led to improvements in the density, spatial and temporal
resolution of hard X-ray tomographic microscopy. For
example, recent advances in X-ray nano-imaging and nano-
tomography (for example, microscopy with X-ray focusing
optics, and X-ray difraction imaging techniques such as
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ptychography), have allowed researchers to investigate and
image objects on a smaller scale.
Rapid technological and computational advances are
driving the feld of HRXCT towards greater usability. Based
upon the increasing number of publications employing
HRXCT in various disciplines that examine a range of
materials, this trend is predicted to continue with HRXCT
becoming a standard imaging technique in the near future
(Cnudde and Boone, 2013). Digital post-processing of data
for analysis and interpretation is also becoming faster and
easier to perform, particularly for non-specialists (Friis
et al.
,
2014). Software generates 3-dimensional views and enables
analyses of the specimens along with performing slices, or
cross-sections, to be taken in any orientation on the same
sample; a feature that is often physically impossible using
traditional microscopy methods (McElrone
et al.
, 2013).
Thus, we are now able to analyze internal features and 3D
volumes of specimens using dedicated software packages
(Cnudde and Boone, 2013), to measure features as small as
soybean pore size (Zong
et al.
, 2017), and to digitally unravel
charred papyrus scrolls from Herculaneum (Bukreeva
et al.
,
2016), research that until recently has not been achievable.
A signifcant drawbank of this technique is the radiation
damage that can results from the high fux of the beam
(Bertrand
et al.
, 2012). This can be minimized by various
measures such that synchrotron tomography is commonly
regarded as non-destructive as the samples under investigation
can normally be re-analyzed using complementary analysis
techniques after the synchrotron experiment (Bertrand
et al.
,
2012). The current study experimentally tested synchrotron
radiation absorption and its efect upon traditional
radiocarbon dating results to test whether or not this is a non-
destructive process at the atomic level. We radiocarbon dated
several charred soybeans from the same two archaeological
Figure 5.
Images captured from Beamline
I13-2 at Diamond Light Source. Charred
Archaeological specimens. a) lentil b)
horsegram c) soybean d)
Chenopodium
sp.
Diameter of the CryoPin is 0.64 mm.
Table 1.
List of Carbonised Soybeans from the Ying Valley, China with direct AMS radiocarbon dates.
SiteLab NoTaxaImagedUncalibrated C
14
date ±30 BPCalibratedage range (95.4%)
Yuanqiao491579SoybeanYes4400
3100–2910 BC
Yuanqiao
491578
SoybeanNo42002900–2670 BC
Yuanqiao
491580
SoybeanNo
43603090–2900 BC
Yuanqiao
491585
SoybeanNo
43903100–2910 BC
Xiawu
491582
SoybeanYes4100
2870–2500 BC
Xiawu
491586
SoybeanYes
43803090–2910 BC
Xiawu
491583
SoybeanNo
3710
2200–2020 BC
Xiawu
491584
SoybeanNo
36102040–1880 BC
Xiawu
491581
SoybeanNo
3690
2200–1970 BC
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sites from the Ying Valley, China (Table 1) and found no
statistically signifcant diferences in the dating results. While
damage can occur at the molecular level, this suggests that
synchrotron radiation does not afect or alter specimens at
an atomic level (Figure 6). The sites of Yuanqiao and Xiawu
are both located in Henan Province, in the Chinese Central
Plains, home of the earliest Chinese States developing in
the second millenium BC. The Yuanqiao site was occupied
during the mid-Yangshao Neolithic phase (4000–2500 BC),
while Xiawu was occupied later, during the Longshan
Neolithic phase (2500–1800 BC).
Despite the benefts and advancements of this imaging
technique, a mechanical issue that can be a limitation is the
physical size and shape of some objects (large and atypical
objects,
i.e.
paintings and rocks) (Bertrand
et al.
, 2012).
Fortunately, for our study of archaeobotanical samples,
they are all conveniently tiny and lie within the very small
feld of the beamline enabling high resolution imaging (see
Methodology section).
Another practical issue that arises is limited access to
National Synchrotrons. As a comparison, we ran several
archaeological soybean specimens in the UCL micro-CT
scanner. From our micro-CT experience we encountered
roughly the same time, equipment use and computer
processing issues with a slightly lower level of resolution for
our archaeobotanical samples when compared with HRXCT.
Recent work on micro-CT has demonstrated burnt-out
chaf temper within the core of ceramic sherds, providing
reliable morphology for taxonomic identifcation (Barron
et al.
, 2017). However, due to the variety in sample size,
shape and composition, there are no fxed or generally-
accepted protocols which exist for micro-CT scanning to
Figure 6.
The results of the outlier analysis
on the model with both the untreated and
irradiated samples for Xiawu. Irradiated
samples are marked with a *. The light grey
distributions show the simple calibrations
and the darker grey distributions show the
modelled posterior estimates, including the
start and end of the sequence. Also included
are the posterior probabilities of the outlier
analysis (P), showing that all of the dates
have a high probability of belonging to the
sequence.
Figure 7.
The results of the outlier analysis
on the model with both the untreated and
irradiated samples. Yuanqiao samples are
marked with a *. The light grey distributions
show the modelled posterior estimates,
including the start and end of the sequence.
Also included are the posterior probabilities
of the outlier analysis (P), showing that all of
the dates have high probability of belonging
to the sequence.
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date (Cnudde and Boone, 2013). For our samples, we did
fnd the greatest resolution possible with HRXCT to better
address our research questions.
Another factor to be considered is the very large data
fles generated from imaging each specimen using HRXCT.
The processing power needed to analyze the data after each
HRXCT imaging session is currently beyond the scale of
most standard university desktop computers. To help with
this challenge, Diamond Light Source has developed new
cluster-run reconstruction software (Savu, Atwood
et al
.,
2015; Wadeson and Basham, 2016), and I13 has a data
beamline (Bodey and Rau, 2019) for data analysis visits.
However, the use of Savu, the new tomography data
processing tool developed by Diamond Light Source
to compile the large image fles, and its continuous
improvements in functionality, is advancing quickly (http://
www.diamond.ac.uk/Science/Computing/Data-Analysis.
html; savu.readthedocs.io/en/late; Basham
et al.
, 2015;
Atwood
et al.
, 2015; Wadeson and Basham, 2016). Despite
the challenges of handling and analyzing large datasets,
the benefts of HRXCT to achieve 3-dimensional, high-
resolution images, as well as the possibilities of addressing
novel research questions – particularly in a non-destructive
and non-invasive manner in intact samples – makes the
eforts worth it.
Another major critique of this nascent feld is that until fairly
recently the various heritage and archaeological disciplines
have largely worked in isolation of each other and hence
“the feld in some respects still lacks the strong collaborative
efort that was put in place in other felds of synchrotron
studies such as for structural biology and nano-sciences”
(Bertrand
et al.
, 2012). However, as more interdisciplinary
studies are undertaken using imaging technology on various
beamlines, with the support of National Synchrotrons, this
feld is becoming increasingly more robust.
1.3 Case study – seed coat thickness
One of the shared domestication traits in many dicotyledonous
crop species, notably in the bean family (Fabaceae), is a
thinning of the seed coat or testa associated with changes
in germination towards faster and more uniform seed
germination (Butler, 1989; Gepts, 1998; Fuller and Allaby,
2009; Fuller
et al.
, 2014). However, documenting this trait in
archaeological seeds has been limited, because observations
of the seed coat requires the destructive process of sectioning
seeds in half and then it is difcult to achieve a perpendicular
view with an SEM. Thus, there is a limited possibility to
document systematically the variation in this trait across all
parts of the seed coat.
To investigate this issue of seed-coat thickness as a trait of
domestication, we applied for and were awarded time on the
Diamond-Manchester Imaging Branchline I13-2 (Rau
et al.
,
2011; Pesic
et al.
, 2013) for our study of archaeobotanical
material. Through HRCXT, we found that seeds and
their seed coats could be imaged in their entirety, non-
destructively, allowing assessment of variation in the seed-
coat thickness over the entire circumference of the seed. We
explored this methodology using specimens of the native
Indian pulse crop horsegram (
Macrotyloma uniforum
) from
archaeological levels in South Indian sites Hallur, Piklihal,
Sanganakallu, and Paithan, dating between 1900 BC and AD
500, thus providing a chronological sequence that captures
much of the domestication process in this crop (Murphy and
Fuller, 2017).
As previously mentioned, high energy synchrotron X-rays
are able to penetrate through thicker materials, providing
a tool for non-destructive examination of features such as
the interior structure of porous materials, as for example in
soybean (
Glycine max
) (Zong
et al.
, 2017). An additional
beneft of our study was the ability to visualize and quantify
the internal structures and preservation of both horsegram
and soybean at the single cell level (Video 1). In both
taxa we were surprised at the level of porosity of charred
archaeological specimens (Figure 4) (Fuller and Murphy,
2016; Murphy and Fuller, 2017). We suspect that this is due
to the oily composition of soybean and its response to the
charring process. Whereas Zong
et al.
(2017) have suggested
that the variation in internal structure in archaeological
soybeans is due to difering proportions of oil vs protein,
we suspect that the impact of charring and preservation
conditions in structurally-preserved voids requires further
investigation (Fuller, 2017).
2. Methodology
2.1 Sample preparation and data collection
Archaeological and modern seed specimens were
individually mounted with clear nail varnish on the end of
18mm CryoPins (Molecular Dimensions Limited, MD7-410)
and mounted on CryoCaps (MD7-400) at I-13-2 (Figure 3).
Images were captured with the pco.4000 detector and pco.
edge 5.5 detectors for the two beamlines, respectively. These
were mounted on visible light microscopes of variable
magnifcation, coupled to scintillators to convert X-rays
to visible light. With the pco.edge 5.5 (which we used for
pink beam imaging), 8× total magnifcation was sufcient to
allow the imagining of whole seeds, giving a feld of view
of 2.1×1.8 mm and an efective pixel size of 0.8125 μm
(Murphy and Fuller, 2017). Samples were continuously
rotated through 180 degrees during data collection with
exposure times of 70 ms. The propagation distance was set
to 20 mm to give a low level of phase contrast. The undulator
gap was set to its minimum position of 5mm and the beamline
was fltered with 1.3 mm pyroltyic graphic, and 3.2 mm
aluminium. For each scan “fat feld” and “dark feld” images
were recorded. Flat feld images are those images without
the sample in the beam. These are often collected before
and after the scan of the sample by horizontally translating
the sample. Dark felds are collected by closing the X-ray
shutter which measures the signal the camera produces with
no X-rays (McElrone
et al.
, 2013).
The number of projections taken during the rotation can
have a signifcant impact on the size of the raw dataset,
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length of the scan, and fnal image quality, but there are
diminishing returns in terms of quality which should be
taken into consideration. We trialled a variety, and settled
upon 6001 projection images (67 GB with dark and
fatfelds). In terms of data storage this may present an issue
of concern to some users, although reconstructed volumes
are smaller, at 51 GB for the pco.edge. Segmentations
(classifcation of voxels into diferent components of a
specimen) can be such a bottleneck that best-quality data
are key to project efciency. Thus, there is always a balance
to be struck between acquisition time and image quality
(Cnudde and Boone, 2013). Hence, for our own samples,
we decided to have a larger dataset size and improved
visual resolution for our samples as we were interested
in the smaller subset of seed coat thickness. In total, the
majority of our samples took 20–30 minutes to image in
their entirety using the above specifcations.
Tomographic reconstruction (generation of 3D volumes
from 2D images) was computed with DAWN (Basham
et al.
,
2015; Titarenko
et al.
, 2010) and Savu run on the Diamond
Light Source computing cluster for our frst and second
beamtimes, respectively. Savu is a modular pipeline which
facilitates various for artefact corrections, including zinger
removals, dark and fatfeld correction, optical distortion
corrections and ring artefact suppressions Strotton
et al.
2018; Vo
et al.
, 2018; 2015). Imaging produced between
2000 and 2672 thin section slices across each seed. Each slice
was approximately 25 MB and the combined tomographic
image dataset per seed was up to 63 GB (Figure 8; Video 1).
When these thin slices are added together with the raw scan
images, the storage space for the basic image data for each
seed was of the order of 150 GB. This tomographic thin
section can provide highly accurate measurements of seed
coat thickness and the entire circumference of the slices,
Figure 8.
a) Archaeological soybean from
China (YUQP2H2, Diamond Light Source
87435). b) Archaeological
Chenopodium
sp. from the site of Haimenkou (HMK,
Diamond Light Source Lab number 87469).
c) Archaeological lentil from Jarmo,
Kurdistan (30/MN 14, Diamond Light
Source 87467). d) Archaeological horsegram
from Hallur, India with an intermediate
thickness seed coat (HLR98A4, Diamond
Light Source 70027). e) Archaeological
horsegram from Paithan, India with a thin
seed coat (PTN721, Diamond Light Source
70053).
Figure 9.
a) Image of archaeological
soybean from Baiyangcun (BYC H118,
Diamond Light Source 87435). b) The same
archaeological soybean segmented using
SuRVoS software at Diamond Light Source.
February 2018. The yellow is the testa or
seedcoat, the red is the endosperm, the blue
is the empty space or voids present in the
interior of the seed volume, and the purple
is a protein pore.
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and multiple slices can be measured. In order to visualize
our samples in 3D, we resampled the slices using the open
source image processing package Fiji (Schindelin
et al.
,
2012) into smaller, lower bit depth slices and visualized
them in Avizo (FEI Systems, Inc., USA). Using this it is
easy to visually separate the internal cellular tissue and
endosperm from the seed coat but, due to similarities in
densities, it is much harder to segment them using standard
image segmentation algorithms. Segmentation refers to
partitioning an image into separate meaningful segments; in
our case, the seed coat and endosperm (see Figure 6). To do
this, we used the recently-developed open-source software
Super Region Volume Segmentation (SuRVoS) developed
by Diamond and University of Nottingham (Luengo
et al.
,
2017), which takes a machine learning approach to volume
segmentation. Firstly, it includes a number of image flters
that can reduce noise and enhance features. This pre-
processing stage is important to establish a representation of
the 3D data that aggregates voxels into supervoxels based on
edges, thus lowering the total number of units to deal with,
as well as enhancing the dissimilarity between the regions to
be segmented. Secondly, it uses model training techniques,
where the user annotates, using diferent coloured brushes,
super-regions that correspond to the diferent segments.
These annotated regions become part of the algorithm
training dataset, which is then used to predict the full extent,
in the full 3-dimensional volume, of those segments (Figure 9
Y-axis thickness in microns; Video 2). Once segmentation
has been accomplished, it is possible to quantify the target
structural or functional changes in texture, and morphology
– including volume, length or width of individual segments
(Cnudde and Boone, 2013).
2.2 Theories and reasoning
As mentioned previously, seed coat thickness, particularly
in pulses such as pea (
Pisum sativum
) (Weeden, 2007),
lentil (
Lens culinaris
) (Ladizinsky, 1985; 1987), horsegram
(Murphy and Fuller, 2017) and soybean (Lee
et al.
, 2011),
is a classic trait of domestication. The null hypothesis, that
there is no change over time in seed coat thickness, was
rejected. Our results support the view that, because of human
intervention leading to domestication, there were genetic and
phenotypic changes in which seed coat thickness decreased
through time allowing germination and water penetration to
occur more rapidly; an advantage in plants under cultivation
(Fuller and Allaby, 2009; Butler, 1989).
3. Results
We rejected the null hypothesis of no change over time
towards decreasing seed coat thickness in horsegram
(Figures 5a; 5b; 6; 7). Instead we demonstrated that average
seed coat thickness gradually became thinner over time. The
results of our beamtime demonstrate that it is possible to
non-destructively image archaeological seeds using HRXCT
with high intensity, highly coherent synchrotron radiation
with high spatial resolution with good signal to noise ratio,
phase contrast, FOV (Murphy and Fuller, 2017).
These results represent the frst time archaeological seed
coats were measured throughout the entire circumference
of the seed. Our results demonstrated empirically that there
is considerable variation within individual archaeological
specimens in seed coat thickness and we can confdently
conclude that single spot estimates of testa thickness may
Figure 10.
Boxplot of archaeological
testa thickness measured on archaeological
specimens of diferent ages (after Murphy
and Fuller, 2017) Y-axis thickness is in
microns.
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be misleading (Figure 6). An unexpected bonus was that we
were able to assess the extent of internal tissue preservation
of archaeological seeds through HRCXT (Figure 5c).
4. Discussion
The HRXCT data generated for horsegram indicate that we
were able to document evidence for morphological change in
terms of average seed coat thickness over time in India. We
acknowledge that some of this testa thickness variation may be
due to diferential shrinkage rates during preservation/charring
on diferent parts of the same seed and post-charring damage
leading to apparent thinning. This points to the need for larger
numbers of testa thickness observations. Importantly, we
found that there was considerable variation within individual
specimens. However, between-specimen variation indicates
temporal trends over time in terms of phenotypic and genetic
changes in this taxon (Murphy and Fuller, 2017).
Our results for seed testa thickness in horsegram reveals a
stepwise thinning over time with domestication, which would
suggest that this trait is under the control of at least two or
more genes (Figure 7). We therefore reject the Abbo
et al.
(2014, p.351) thesis that it is only the domestication traits
which show “a clear domesticated-wild dimorphism [which]
represents the pristine domestication episode, whereas
traits showing a phenotypic continuum between wild and
domesticated gene pools mostly refect post-domestication
diversifcation”. Based upon the time depth of our samples,
dating from the Neolithic to the Early Historic period in
South Asia, we can be fairly confdent that our archaeological
samples captured much of the “domestication episode” in
horsegram domestication (Murphy and Fuller, 2017). These
early archaeological samples of horsegram are far too early
to represent the “post-domestication” evolution of this crop
in South Asia. Furthermore, this contradicts the hypothesis of
“domestication before cultivation” proposed for
Lens culinaris
(Ladizinsky, 1987), which if true ought to apply to many or
all pulse domestications. Instead seedcoat thinning evolved
gradually over several centuries, up to a millennium in the
case of
Macrotyloma uniforum
(Murphy and Fuller, 2017).
Higher-resolution study focusing on morphometric
and structural changes associated with domestication of
archaeobotanical remains can be used in conjunction with,
and combined with, previous traditional measurements
(Fuller, Colledge, Murphy and Stevens, 2017; Fuller and
Murphy, 2018). We intend to continue this successful line of
investigation into seed coat thinning as a trait of domestication
in other archaeological taxa from the Old World. We expect
that the trends will be similar, with a decrease seen with
domestication over time with gradual step-wise change.
5. Conclusions
HRXCT provides a useful technique for non-destructive
investigations and ofers new opportunities to investigate
the cellular and structural properties within archaeological
samples and, as this paper has demonstrated, archaeobotanical
samples in particular. This non-destructive and non-invasive
technique allows 3-dimensional imaging and more advanced
and accurate morphometric measurements of internal
features and/or structures that had hereto been impossible to
visualize and quantify (Friis
et al.
, 2014). While the study
of seed coat thinning during domestication has been carried
out by SEM based examination of a few point measurements
on each seed (
e.g.
Fritz
et al.
, 2017), synchrotron images
can provide a more statistically robust assessment of within-
specimen variation in this trait and requires neither specimen
destruction, nor the use of cracked or broken specimens. This
technology, although currently somewhat limited in terms
of time and access to beamtimes at National Synchrotrons,
ofers simple, non-destructive preparation of samples and
exciting results. Based upon these two rounds of successful
imaging results, we believe that this new methodology has
wider applications and utility for investigating other aspects
of plant domestication.
Acknowledgements
This research was part of the Comparative Pathways to
Agriculture Project (ComPAg) funded by the European
Research Council (grant 323842) and the Early Rice Project
funded by the UK Natural Environment Research Council
(NE/ N010957/1). Synchrotron imaging was supported by a
grant of beamline time from Diamond Light Source (www.
diamond.ac.uk), grant of beam time MT12082 “Drawing
a thin line: Examining seedcoat thickness in relation to
domestication in
Macrotyloma uniforum
(horsegram)”
and MT1606 “Drawing a thinner line: Examining seedcoat
thickness in relation to domestication in
Macrotyloma
uniforum
(horsegram)”. We acknowledge Diamond Light
Source for time on Branchline I-13-2 and its associated data
beamline (Bodey and Rau, 2017) under Proposal MT12082
and MT16061. We thank Beamline staf and its associated
data beamline (Bodey and Rau, 2017) for their assistance.
We would like to thank Michele Darrow, Imanol Luengo
and Mark Basham for their assistance with SuRVoS software
at Diamond. For access to modern reference materials we
would like to thank the Royal Botanic Gardens, Kew; the
London Natural History Museum herbarium, and USDA
national germplasm collection.
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Websites
http://www.diamond.ac.uk/Beamlines/Materials/I13.html [Accessed 1/09/2017]
http://www.diamond.ac.uk/Beamlines/Materials/Techniques/XTT.html
[Accessed 1/09/2017]
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image/svg+xml
Charlene Murphy, Dorian Q. Fuller, Chris Stevens, Tom Gregory, Fabio Silva, Rita Dal Martello, Jixiang Song, Andrew J. Bodey, Christoph Rau:
Looking Beyond the
Surface: Use of High Resolution X-Ray Computed Tomography on Archaeobotanical Remains
Video 1.
Compiled video of archaeological horsegram imaged during the 2015 session at Diamond Light Source.
Video 2.
3-dimensional render of archaeological soybean from China dating to the Neolithic. Imaged during the 2017 session at Diamond Light Source.
IANSA 2019 ● X/1 ● 7–18
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